Generation of mESC lines stably overexpressing rabbit <i>Hcn4</i>.

<p>A. A transfection construct bearing the rabbit <i>Hcn4</i>-IRES-<i>EGFP</i> cassette. B. Representative living mESCs observed by phase contrast microscopy (a to c) and fluorescence microscopy (d to f). <i>Hcn4/EGFP</i> or <i>EGFP</i>-stably-transfected mESCs (HCN4/EGFP mESC-2 and EGFP mESC-1 cell lines) were positive for EGFP (green) fluorescence (e and f). C. Immunofluorescence staining of a pluripotency marker, OCT4, in <i>Hcn4/EGFP</i>-stably-transfected mESCs (a to c) and <i>EGFP</i>-stably-transfected mESCs (d to f). OCT4 was expressed in both mESC lines. Bar = 50 μm. D. Measurement of <i>I</i>_f currents in <i>EGFP</i>-stably-transfected mESCs (a) and <i>Hcn4/EGFP</i>-stably-transfected mESCs (b). Activation of the <i>I</i>_f current was demonstrated in <i>Hcn4/EGFP</i>-stably-transfected mESCs. <i>I</i>_f currents through activated HCN channels were obtained during hyperpolarizing test pulses of 5 seconds between -45 and -125 mV in 20 mV increments from a holding potential of -35 mV.